Figure 5. REF-1 assay.

(A) HCT116 nuclear extract was incubated with ³²P-labeled consensus (CON) or mutant (MUT) AP-1 oligonucleotide substrates, and binding reactions were resolved on a non-denaturing polyacrylamide gel. Control reactions without nuclear extract (no extract) are shown. The arrow designates the position of the AP-1-specific consensus binding complex, not seen with the MUT double-stranded DNA. Higher molecular weight non-specific complexes are observed. (B) Reduced wild-type (WT) or variant APE1 protein was incubated with HCT116 nuclear extract in the presence of the ³²P-labeled AP-1 CON DNA substrate. Shown is the AP-1-specific complex in the absence (no protein) or presence of the indicated reduced APE1 protein after phosphorimager analysis. Plotted is the relative AP-1 DNA binding activity, in comparison with reduced WT protein. Values represent the average and standard deviation of 3 independent experimental points.